The baculovirus expression vector system (BEVS) is a proven, powerful and versatile method of eukaryotic protein expression. It is used to produce vaccines, diagnostics, and biologically active proteins for a multitude of research projects. Like all expression systems, however, BEVS has its disadvantages. One is the fact that expression is short-lived due to virus-induced cell death and lysis. ParaTechs has already commercialized a product that addresses this shortcoming. Cell lines that express a viral ankyrin gene show delayed death and lysis of baculovirus-infected cells, thereby significantly enhancing recombinant protein production. This activity, referred to as vankyrin-enhanced BEVS (VE-BEVSTM), boosts target protein expression up to 20-fold, depending on the protein and method of vankyrin delivery. A second limitation of baculovirus expression is that insect cells lack the ability to produce terminally sialylated, complex N-glycans, which limits the usefulness of BEVS for the expression of human therapeutic proteins. This proposal addresses that issue and aims to develop new cell lines that produce authentic "humanized" N-glycans in the context of vankyrin-enhanced baculovirus technology. To obtain this goal, three different insect cell lines that have been previously engineered to produce mammalian glycosylation enzymes will be co-infected with baculoviruses that encode a vankyrin gene and different test glycoproteins. The transgenic cell line with the highest vankyrin response will be transformed with two different vankyrin genes under the control of several promoters. Polyclonal cells will be screened for enhanced glycoprotein expression, and the most promising cell population will be cloned. Different monoclonal lines will be tested for production of complex terminally sialylated glycoproteins, as compared to the non-transformed parental line. These cells will provide the highest yields of accurately processed secreted and transmembrane proteins, and will be marketed to individual researchers and pharmaceutical companies engaged in structure-function studies of the human secretome and development of protein therapeutics. These Phase I studies will significantly expand the applications of ParaTechs'vankyrin- enhanced BEVS technology. ParaTechs personnel and the Jarvis group have experience with all of the techniques to be used in these studies and do not anticipate difficulty achieving the objectives. The data derived from funding of this Phase I proposal will position ParaTechs to initiate Phase II studies to demonstrate the utility of these cell lines for production of transmembrane and secreted proteins in collaboration with potential end users. PUBLIC HEALTH RELEVANCE: The inability of insect cells to produce terminally sialylated, complex N-glycans limits the usefulness of baculovirus expression system for the production of human therapeutic proteins. This proposal addresses that deficiency and aims to develop new cell lines that produce authentic "humanized" N-glycans in the context of ParaTechs vankyrin-enhanced baculovirus technology. Successful completion of these objectives will produce cells that provide the highest levels of accurately processed secreted and transmembrane proteins and will be marketed to individual researchers and pharmaceutical companies engaged in structure-function studies of the human secretome and development of protein therapeutics.